New therapeutic strategies are needed to circumvent the toxicity of current anti-HIV drugs and the rapid selection of drug-resistant virus. RNA aptamers targeted to the reverse transcriptase (RT) inhibit viral replication when expressed inside cells, and they offer a potential alternative to small molecule drugs. Aptamers are hypothesized to be less susceptible escape mutations than small molecule drugs. The long-term goal of this project is to define the influence of RT amino acid sequence variations-both natural and evolved-on RT function and on inhibition by nucleic acid aptamers. In Aim 1, we will clone, express and purify RT from several strains, emphasizing the major HIV-1 groups and subtypes. DNA polymerization, RNaseH and strand transfer activities will be measured for each RT in challenges from a panel of inhibitory RNA aptamers, with and without nucleocapsid NCp7. Aim 2 will define the amino acids that confer aptamer resistance in viral challenges of human cells, and will establish whether in vitro biochemical parameters correlate with intracellular behavior. This aim constitutes a major new initiative for our research program. Aim 3 will determine whether multi-clade targeting during aptamer selection enhances cross-reactivity with RT from clades that were not initially targeted during the selection. We will generate new RNA aptamers that inhibit pairs or sets of RT at increasing evolutionary distances from HXB2. Aptamers that bind diverse RT are hypothesized to recognize highly conserved features that are less subject to aptamer resistance mutations, making them especially valuable. The results from this project will establish in vitro the molecular basis for variable sensitivities to aptamer challenges previously observed in cell-based viral assays, they will begin to measure RT's capacity to evolve escape from inhibition, and they will generate new aptamers that are less subject to escape mutations. [unreadable] [unreadable] [unreadable]